The long-term objectives of this grant are to increase understanding of the mechanisms whereby cultured cells and tumors change genetic properties by the process of gene amplification, predominately with studies on resistance to methotrexate. The studies to be undertaken include: 1) an analysis of the nucleotide sequences constituting promoter regions of the dihydrofolate reductase gene, and studies on the synthesis and metabolism of the dihydrofolate reductase mRNA in the cell cycle; 2) an analysis of the propensity of normal, malignant, and metastatic breast epithelium to undergo spontaneous or induced amplification of the dihydrofolate reductase gene as detected by use of the fluorescence activated cell sorter and molecular biology technology; 3) an analysis of resistance to methotrexate that is associated with an unstable phenotype and increased ability to bind methotrexate at 4 degrees, and which has the properties of an altered transport of methotrexate, employing a combination of techniques to characterize the putative overproduced protein and its gene; 4) an attempt to generate resistance to radiomimetic drugs in such a fashion as to select for cells in which there is an increased capability for DNA recombination which results in the resistance phenotype by a gene amplification mechanism.